Complete hydatidiform mole (CHM) is a unique disease of gynecological neoplastic conditions that are derived from the placenta.1 It is a trophoblastic disease characterized by androgenetic diploidy. It involves overgrowth of variable amounts of villous trophoblasts and extravillous trophoblasts.2 Complete hydatidiform mole may show invasion into the myometrium but not necessarily. Although progress has been made in understanding the biology of human trophoblasts and their pathological mechanism, the pathogenesis of CHM is still not completely understood, due in part to the increasing rarity of the disease and the lack of an animal model.
A new class of small noncoding RNA molecules of about 22 nucleotides long, called microRNAs (miRNAs), plays important regulatory roles in protein translation and mRNA degradation.3 MicroRNAs act mainly by base pairing to the 3′-untranslated regions of mRNAs. MicroRNA activity is important in the regulation of physiological and pathological processes, including development, cell proliferation/growth and differentiation, angiogenesis, apoptosis, inflammation, oncogenesis, and other processes in endothelial cells.3,4 Recent studies showed that a total of 8 miRNAs, including miR-512-3p, miR-517a, miR-517b, miR-518b, miR-519a, miR-1185, miR-1283, and miR-1323, are preferentially expressed in the placenta. In other words, there are 8 placenta-associated miRNAs.5
It is possible that these 8 placenta-associated miRNAs may be involved in the functional regulation of trophoblastic cells during the first trimester. Unfortunately, there is so far no report on this possibility. The goal of this study was to identify the differential expression of placenta-associated miRNAs in trophoblastic cells in CHM versus normal pregnancy, using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization (ISH).
MATERIALS AND METHODS
Human placentas were obtained following approved protocols by the Medical Ethics Committee of the China Medical University Shengjing Hospital.
We collected placental tissues from first-trimester elective termination of normal pregnancies (control group, 20 cases) and patient with CHM (CHM group, 12 cases, at the time of therapeutic abortion). The clinical characteristics of these 2 groups are shown in Table 1. There were no significant differences in maternal age and gestational age between the 2 groups.
Inclusion criteria for the 20 cases in the control group were the following: (1) no symptoms, signs or history of threatened abortion, with normal findings by ultrasound, and elective abortion at gestational age of 6 to 13 weeks in our birth control clinic; (2) regular maternal menstrual period of 25 to 35 days, no other systemic diseases, and no history of exposure to hazardous substances; (3) anticardiolipin antibody, antinuclear antibody, anti–double-stranded DNA antibody, immunoglobulin M antisperm antibody, and immunoglobulin G antibody in maternal blood were all negative; (4) rubella virus, cytomegalovirus, herpes virus, Toxoplasma gondii immunoglobulin M in maternal blood were all negative; (5) transvaginal ultrasound showing no anatomical abnormalities of the reproductive system; (6) normal chromosome G-banding karyotype analysis of the parents and no abnormal findings in fluorescent ISH and morphology analysis of chromosomes 13, 16, 18, 21, 22, X, and Y; and (7) routine computer-aided semen analysis of the father showing normal results, and sperm staining that revealed an abnormal sperm rate of more than 70%.
Inclusion criteria for the 12 cases in the CHM group were the following: B-ultrasound test suggesting CHM, pathology of the removed tissues confirming diagnosis of CHM, no preoperative chemotherapy, regular postoperative follow-up showing that blood β-HCG level returned to normal within 8 weeks, and vesicular mole tissues of about 1 cm3 in size.
Tissues of the 2 groups were processed immediately after collection (within 20 minutes), and 32 samples were used for quantitative real-time PCR analysis.
Each sample of chorionic villous tissue was washed extensively with normal saline within 5 minutes of extraction. One tissue block of about 0.5 × 0.5 × 0.5 cm was quickly placed in lysis buffer for RNA extraction, followed by quantitative real-time PCR analysis, to determine the expression level of these 8 placenta-associated miRNAs in placenta tissue. The remaining tissue block was placed in cryopreservation tubes, quickly submerged in liquid nitrogen, and transferred to a −80°C freezer the next day.
MiRNA Isolation, Reverse Transcription, and Real-Time Quantitative PCR
Total RNA containing small RNA molecules was extracted from 0.5 × 0.5 × 0.5-cm portions of chorionic villous tissue with the mirVana miRNA Isolation kit (Ambion/Applied Biosystems) according to the manufacturer’s instructions. Quality assessment and concentration measurements of total RNA were performed with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Only samples with a 260/280 absorbance ratio of greater than 1.8 and less than 2.1 were used.
We subjected 50 ng total RNA to reverse transcription using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions.
Quantitative real-time reverse transcription–PCR was performed with gene-specific fluorescent TaqMan probes using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems), SDS 2.3 software, and RQ Manager (Applied Biosystems), according to the manufacturer’s instructions. We selected RNU6–2 (also known as U6) as the endogenous control, because of its stable expression among samples. The probes used for miR-512-3p (4381034), miR-517a (4373243), miR-517b (4373244), miR-518b (4373246), miR-519a (4373249), miR-1185 (4373249), miR-1283 (4373249), miR-1323 (4373249), and RNU6–2 (4373381) were from ABI (TaqMan MicroRNA Assays; Applied Biosystems). We then performed quantitative PCR with the TaqMan PCR Master Mix (4369016; Applied Biosystems). Each sample with each probe was analyzed in triplicate. MicroRNA expression was quantified by the–ΔΔCT method.
Using the same total RNA of these 32 samples, we repeated the experiment twice with the same procedure.
Target Prediction of miRNAs
MiRGen (http://www.diana.pcbi.upenn.edu/miRGen.html), miRANDA (http://www.microrna.org/), PicTar (http://pictar.bio.nyu.edu/), miRNAMap (http://mirnamap.mbc.nctu.edu.tw/), and TargetScan 4.0 (http://www.targetscn.org/) were used to predict the target genes of the differentially expressed miRNAs.
In Situ Hybridization
In situ hybridization was performed using miRCURY locked nucleic acid detection probes (Exiqon, Vedbaek, Denmark) for miR-517a, miR-517b, miR-518b, and miR-519a.
Briefly, formalin-fixed, paraffin-embedded human placental tissue samples were deparaffinized in xylene and treated with proteinase K (15 μg/mL; Wako) at 37°C for 10 minutes and then hybridized for 1 h at 55°C with hybridization buffer containing 50 nM 3′-digoxigenin–labeled locked nucleic acid miRNA probes. After hybridization, the sections were incubated with sheep anti–digoxigenin–alkaline phosphatase–Fab fragments (1:2000 dilution; Roche, Basel, Switzerland) at room temperature for 1 hour and then visualized with nitroblue tetrazolium chloride/5-bromo-4-chloro-3′-indolyl p-toluidine salt (NBT/BCIP; Roche) at 30°C for 2 h. After visualization of the ISH signal, the slides were counterstained with 4′, 6-diamidino 2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) and then mounted in aqueous mounting medium (Pristine Mount Research Genetics, Huntsville, AL).
Data were analyzed with SPSS16.0 software (SPSS Inc, Chicago, IL) and are shown as the mean (SD). The threshold cycle (CT) reflects the cycle number at which the fluorescence that is generated in the reaction crosses a preselected threshold and is inversely related to the concentration of cDNA before amplification. MicroRNA expression was analyzed by the comparative CT method.
Differences in miRNA expression (ΔCT) between the groups were evaluated using an independent-samples t test using SPSS 16.0 for Windows (SPSS 16.0). If the P value of the independent-samples t test is less than 0.05, the difference was recognized to be significant. Up-regulation was considered significant if fold change was 1.8 or greater, and down-regulation was considered significant if fold change was 0.55 or less.
MiRNA Expression Levels in Human Placental Villous Tissues
The expression levels of miR-512-3p, miR-517a, miR-517b, miR-518b, miR-519a, miR-1185, miR-1283, and miR-1323 (Fig. 1) in first-trimester placental villi of normal pregnancies were compared with those of CHM. The relative expression of miRNAs was calculated by normalization with U6 small nuclear RNA expression using the comparative CT method.6
Quantitative real-time reverse transcription–PCR detected differentially expressed miRNAs in placentas of the CHM group and the control group, including miR-517a (∼1.8576-fold increase in the CHM group), miR-517b (∼8.4549-fold increase in the CHM group), miR-518b (∼6.6553-fold increase in the CHM group), and miR-519a (∼2.5574-fold increase in the CHM group). The data are summarized in Table 2, and the possible roles of some of these miRNAs are discussed later.
MiRNA Expression Location in CHM
For the 4 miRNAs already described to be differential expression in the 2 groups, miR-517a, miR-517b, miR-518b, and miR-519a, we further investigated their in vivo localization in CHM tissues by ISH. MiR-517b and miR-518b were detected exclusively in the trophoblast layer; little signal (if any) was observed in villous stroma cells or fetal endothelial cells. MiR-517a and miR-519a were expressed not only in the villous trophoblasts but also in some villous stroma cells (Fig. 2).
Hydatidiform mole is characterized by varying intensity of trophoblastic proliferation associated with an absent or abnormal fetus. Partial moles demonstrate identifiable fetal or embryonic tissue with focal edema in the chorionic villi, whereas CHMs undergo a uniform hydatid enlargement of villi in the absence of the fetus. Fifteen percent to 20% of complete moles develop into invasive moles or choriocarcinoma, representing different degrees of invasiveness.
It is well known that human pregnancy is associated with extensive growth and remodeling of the uterus and placenta. Trophoblasts play an important role in establishing pregnancy. A successful human pregnancy requires fetal cytotrophoblasts to adopt tumorlike properties. Cytotrophoblasts attach the conceptus to the endometrium by invading the uterus, and they initiate blood flow to the placenta by breaching maternal vessels. The entry of trophoblastic cells into the maternal endometrium is one of the key events in human placentation.7 However, unlike tumor metastasis, cytotrophoblast invasion is precisely controlled. Thus, normal trophoblast cells are also known as pseudomalignant cells.8
Inadequate or excessive invasion by trophoblasts results in gestational trophoblast diseases, such as preeclampsia, intrauterine growth restriction, hydatidiform moles, and choriocarcinomas.9,10 Complete hydatidiform mole are abnormally developing pregnancies with hyperproliferative vesicular trophoblasts and defective fetal development.2 Normal human pregnancy is accompanied by controlled trophoblast cell proliferation, differentiation, and invasion. However, CHM placentas are overgrown and show varying degrees of local invasion and metastasis.
Research on the expression and secretion of miRNA in human placental trophoblast cells have just begun in recent years. In 2004, investigators found that certain miRNAs were highly expressed in mouse and human placenta, suggesting that miRNA may play some roles in the growth and development of the placenta.11,12 Later, a few additional studies reported the correlation between miRNAs and the function or disease status of the placenta.13,14
In 2009, Luo et al5 determined the expression levels of miRNAs in several different tissues (brain, heart, lung, ovary, testis, placenta, etc) and found 8 miRNAs that were preferentially expressed in the placenta (miR-512-3p, miR-517a, miR-517b, miR-518b, miR-519a, miR-1185, miR-1283, and miR-1323), which became known as placenta-associated miRNAs.5 It is conceivable that the highly expressed placenta-associated miRNAs may be involved in the regulation of invasion, proliferation, and apoptosis of the trophoblastic cells.
Our study detected for the first time the expression levels and locations of these 8 placenta-associated miRNAs in first-trimester placental villi from CHM and normal first-trimester induced abortion. We found that the expression of 4 miRNAs (miR-517a, miR-517b, miR-518b, and miR-519a) was significantly decreased in CHM tissues. Further ISH studies showed that these 4 miRNAs were expressed in trophoblastic cells. Furthermore, miR-517b and miR-518b expressed only in the trophoblast cell layer of the terminal villi, but not in the villous stroma, blood vessels, and so on.
Recently, 1 study reported that miR-517a has a carcinogenic effect in vivo for hepatocellular carcinoma (HCC). In addition, the effect of miR-517a on the 19q13.41 locus as a novel oncogenic miRNA in HCC was evidenced by its ability to promote cell proliferation and migration in vitro and to induce tumorigenesis in vivo.15 MiR-517b was differentially expressed in various tumor tissues including HCC15 and tumor tissues.16 Moreover, miR-518b was also shown to play an important role in the pathogenesis and development of esophageal cancer.17 MiR-519a is not only involved in cell proliferation,18 but is also closely associated with epithelial ovarian cancer.19 Based on the previously mentioned studies, we speculate that miR-517a, miR-517b, miR-518b, and miR-519a may play a role in the pathogenesis of CHM by regulating functions of trophoblastic cells, in a similar way as in tumorigenesis.
This study provided some putative targets for each differentially expressed placenta-associated miRNA between the 2 groups (Table 2). We found that these differentially expressed placenta-associated miRNAs all have corresponding target genes involved in functions of trophoblastic cells. For example, among the target genes of miR-517b, ZEB-2 (zinc finger E-box binding homeobox 2) can enhance cell proliferation through the transforming growth factor β/bone morphogenetic protein signaling pathway, as well as being closely associated with the invasion and apoptosis of breast cancers.20 A study of another target gene of miR-517b, Wnt4 (wingless-type MMTV integration site family, member 4), showed its expression in the adult uterus during pregnancy, indicating that this miRNA may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo.21 Among the target genes for miR-519a, p63 may play a role in the pathogenesis of CHM through its interaction with p53-dependent proliferation and apoptotic activities.22 Another target gene of miR-519a, TGFBR2 (transforming growth factor B receptor 2), has been related to trophoblastic invasion and placental implantation.23 PTEN, a tumor suppressor gene, was found to be involved in tumor proliferation and trophoblastic invasion,24 and MMP2 was associated with trophoblastic invasion, and so on.25 Thus, we speculate that these 4 miRNAs may be involved in the pathogenesis of CHM by regulating the functions of trophoblastic cells through these target genes.
We identified 4 placenta-associated miRNAs (miR-517a, miR-517b, miR-518b, and miR-519a) that were differentially expressed in CHM. Two of them (miR-517b and miR-518b) were preferentially expressed at the sites involving functions of trophoblastic cells in early pregnancy villi. In addition, there are literature reports implying that these 4 miRNAs and/or their target genes may be closely related to CHM. Further studies have been carried out by our group to reveal the function of these identified miRNAs, including confirming the interaction between miRNA and their target genes.
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